Somatostatin-dopamine chimeric analogs

ABSTRACT

Disclosed is a series of somatostatin-dopamine chimeric analogs which retain both somatostatin and dopamine activity in vivo. An example is: 6-n-propyl-8β-ergolinglmethylthioacetyl-D-Phe-c-(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2

BACKGROUND OF THE INVENTION

[0001] The present invention is drawn to somatostatin-dopamine chimericanalogs.

[0002] Dopamine is a catecholamine neurotransmitter that has beenimplicated in the pathogenesis of both Parkinson disease andschizophrenia. Graybiel, et al., Adv. Neurol. 53, 17-29 (1990);Goldstein, et al., FASEB J. 6, 2413-2421 (1992); Olanow, et al., Annu.Rev. Neurosci. 22, 123-144 (1999). Egan, et al., Cuff. Opin. Neurobiol.7, 701-707 (1997). Dopamine and related molecules have been shown toinhibit the growth of several types of malignant tumors in mice, andthis activity has been variously attributed to inhibition of tumor-cellproliferation, stimulation of tumor immunity or effects on melaninmetabolism in malignant melanomas. Wick, M. M., J. Invest. Dernnatol.71, 163-164 (1978); Wick, M. M., J. Natl. Cancer Inst. 63, 1465-1467(1979); Wick, M. M., Cancer Treat Rep. 63, 991-997 (1979); Wick, M. M.,Cancer Res. 40, 1414-1418 (1980); Wick, M. M., Cancer Treat. Rep. 65,861-867 (1981); Wick, M. M. & Mui, J. Natl. Cancer Inst. 66, 351-354(1981); Dasgupta, et al., J. Cancer Res. Clin. Oncol. 113, 363-368(1987); Basu, et al., Endoctine 12, 237-241 (2000); Basu, et al., J.Neuroimmunol. 102, 113-124 (2000). Recent studies demonstrated thepresence of D2 dopamine receptors on endothelial cells. Ricci, et al.,J. Auton. Pharmacol., 14, 61-68 (1994); Bacic, et al., J. Neurochem. 57,1774-1780 (1991). Dopamine has recently been reported to strongly andselectively inhibit at non-toxic levels the vascular permeabilizing andangiogenic activities of VPF/VEGF. Basu et al., Nat. Med. 7 (5), 569-574(2001).

[0003] Somatostatin (SS), a tetradecapeptide discovered by Brazeau etal., has been shown to have potent inhibitory effects on varioussecretory processes in tissues such as pituitary, pancreas andgastrointestinal tract. SS also acts as a neuromodulator in the centralnervous system. These biological effects of SS, all inhibitory innature, are elicited through a series of G protein coupled receptors, ofwhich five different subtypes have been characterized (SSTR1-SSTR5)(Reubi J C, et al., Cancer Res 47: 551-558, Reisine T, et al., EndocrineReview 16: 427-442, Lamberts S W, et al., Endocr Rev 12: 450-482, 4Patel Y C, 1999 Front Neuroendocrinology 20: 157-198). These fivesubtypes have similar affinities for the endogenous SS ligands but havediffering distribution in various tissues. Somatostatin binds to thefive distinct receptor (SSTR) subtypes with relatively high and equalaffinity for each subtype.

[0004] There is evidence that SS regulates cell proliferation byarresting cell growth via SSTR1, 2, 4, and 5 subtypes (Buscail L, etal., 1995 Proc Natl Acad Sci USA 92: 1580-1584; Buscail L, et al., 1994Proc Natl Acad Sci USA 91: 2315-2319; Florio T. et al., 1999 MolEndocrinol 13: 24-37; Sharma K, et al., 1999 Mol Endocrinol 13: 82-90),or by inducing apoptosis via SSTR3 subtype (Sharma K, et al., 1996 MolEndocrinol 10: 1688-1696). SS and various analogues have been shown toinhibit normal and neoplastic cell proliferation in vitro and vivo(Lamberts S W, et al., Endocr Rev 12: 450-482) via specific SS receptors(SSTR's) (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198) andpossibly different postreceptor actions (Weckbecker G, et al., PharmacolTher 60: 245-264; Bell G I, Reisine T 1993 Trends Neurosci 16: 34-38;Patel Y C, et al., Biochem Biophys Res Commun 198: 605-612; Law S F, etal., Cell Signal 7:1-8). In addition, there is evidence that distinctSSTR subtypes are expressed in normal and neoplastic human tissues (9),conferring different tissue affinities for various SS analogues andvariable clinical response to their therapeutic effects.

[0005] Binding to the different types of somatostatin receptor subtypeshave been associated with the treatment of various conditions and/ordiseases. (“SSTR2”) (Raynor, et al., Molecular Pharmacol. 43:838 (1993);Lloyd, et al., Am. J. Physiol. 268:G102 (1995)) while the inhibition ofinsulin has been attributed to the somatostatin type-5 receptor(“SSTR5”) (Coy, et al. 197:366-371 (1993)). Activation of types 2 and 5have been associated with growth hormone suppression and moreparticularly GH secreting adenomas (Acromegaly) and TSH secretingadenomas. Activation of type 2 but not type 5 has been associated withtreating prolactin secreting adenomas. Other indications associated withactivation of the somatostatin receptor subtypes include inhibition ofinsulin and/or glucagon for treating diabetes mellitus, angiopathy,proliferative retinopathy, dawn phenomenon and nephropathy; inhibitionof gastric acid secretion and more particularly peptic ulcers,enterocutaneous and pancreaticocutaneous fistula, irritable bowelsyndrome, Dumping syndrome, watery diarrhea syndrome, AIDS relateddiarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitisand gastrointestinal hormone secreting tumors; treatment of cancer suchas hepatoma; inhibition of angiogenesis; treatment of inflammatorydisorders such as arthritis; retinopathy; chronic allograft rejection;angioplasty; preventing graft vessel and gastrointestinal bleeding. Itis preferred to have an analog which is selective for the specificsomatostatin receptor subtype or subtypes responsible for the desiredbiological response, thus, reducing interaction with other receptorsubtypes which could lead to undesirable side effects.

[0006] Somatostatin (SS) and its receptors (SSTR1 to SSTR5) areexpressed in normal human parafollicular C cells and medullary thyroidcarcinoma (MTC). MTC is a tumor originating from thyroid parafollicularC cells that produces calcitonin (CT), somatostatin, as well as severalother peptides (Moreau J P, et al., Metabolism 45 (8 Suppl 1): 24-26).Recently, Mato et al. showed that SS and SSTR's are expressed in humanMTC (Mato E, et al., J Clin Endocrinol Metab 83: 2417-2420). It has beendocumented that SS and its analogues induce a decrease in plasma CTlevels and a symptomatic improvement in MTC patients. However, until nowthe antiproliferative activity of SS analogues on tumor cells had notbeen clearly demonstrated (Mahler C, et al., Clin Endocrinol 33: 261-9;Lupoli G, et al., Cancer 78: 1114-8; Smid W M, et al., Neth J Med 40:240-243). Thus, development and assessment of SSTR subtype analoguesselective on MTC cell growth provides a useful tool for clinicalapplication. Until now, no data concerning specific SSTR subtypeinvolvement in MTC cell growth regulation have been reported.

SUMMARY OF THE INVENTION

[0007] The present invention is concerned with the discovery of a seriesof somatostatin-dopamine chimeric analogs that retain both somatostatinand dopamine activity in vivo, including several of which displayenhanced biological activity over the native somatostatin and dopamineanalogs alone, and the therapeutic uses thereof.

[0008] In one aspect the invention features a dopamine-somatostatinchimer of formula (I).

[0009] wherein:

[0010] X is H, Cl, Br, I, F, —CN, or C₁₋₅ alkyl;

[0011] R1 is H, C₁₋₄ alkyl, allyl, alkenyl or —CN;

[0012] R2 and R3, each are, independently H or absent, provided thatwhen R2 and R3 are absent a double bond is present between the carbonatoms to which they are attached;

[0013] R4 is H or —CH₃;

[0014] Y is —O—, —C(O), —S—, —S—(CH₂)s-C(O)—, —S(O)—, —S(O)₂—, —SC(O)—,—OC(O)—, —N(R5)-C(O)—, or —N(R6)-;

[0015] R5, R6, R7 and R8 each is, independently, H or C₁₋₄ alkyl;

[0016] R6 is H or C₁₋₄ alkyl;

[0017] m is 0 or1;

[0018] n is 0-10;

[0019] L is —(CH₂)p-C(O)—, when Y is —S—, —S(O)—, —S(O)₂—, —O— or—N(R6)-;

[0020] L is —C(O)—(CR7R8)q-C(O)—, when Y is —N(R6)-, —O—, or —S—;

[0021] L is -(Doc)t-, when Y is —C(O)—, SC(O)—,—OC(O)—, —S—(CH₂)s-C(O)—,or —N(R5)-C(O)—;

[0022] p is 1-10;

[0023] q is 2-4;

[0024] s is 1-10;

[0025] t is 1-10; and

[0026] Z is somatostabn analog,

[0027] or a pharmaceutically acceptable salt thereof.

[0028] In another aspect the invention features a dopamine-somatostatinchimer of formula (II),

[0029] wherein:

[0030] X is H, Cl, Br, I, F, —CN, or C₁₋₅ alkyl;

[0031] R1 is C₁₋₄ alkyl, H, allyl, alkenyl or —CN;

[0032] R2 and R3, each are, independently H or absent, provided thatwhen R2 and R3 are absent a double bond is present between the carbonatoms to which they are attached;

[0033] R4 is H or —CH₃;

[0034] R5 is C1-5 alkyl group, or a group of the formula of—(CH₂)rN(CH₃)q;

[0035] Y is —O—, —C(O)—, —S—, —SC(O)—, —OC(O)—, —N(R6)-C(O)—, —N(R7)-,or —N(R8)-(CH₂)s-C(O)—;

[0036] R6, R7, R8, R9 and R10 each is, independently, H or C₁₋₅ alkyl;

[0037] L is —(CH₂)p-C(O)—, when Y is —S—, —O— or —N(R7)-;

[0038] L is —C(O)—(CR9R10)q-C(O)—, when Y is —N(R7)-, —O—, or —S—;

[0039] L is -(Doc)t-, when Y is —C(O)—, SC(O)—, —OC(O)—,—N(R8)-(CH2)s-C(O)—, or —N(R6)-C(O)—;

[0040] m is 0 or 1;

[0041] n is 2-10;

[0042] r is 1-8,;

[0043] q is 2-4;

[0044] p is 1-10;

[0045] s is 1-10;

[0046] t is 1-10; and

[0047] Z is somatostatin analog

[0048] or a pharmaceutically acceptable salt thereof.

[0049] In one embodiment the invention features a compound according tothe formula:

[0050] or a pharmaceutically acceptable salt thereof.

[0051] In another embodiment the invention features a compound accordingto the formula:

[0052] or a pharmaceutically acceptable salt thereof.

[0053] In one aspect the invention features a method of eliciting adopamine receptor agonist effect in a subject in need thereof, whereinsaid method comprises administering to said subject an effective amountof a compound according to formula (I) or formula (II), or apharmaceutically acceptable salt thereof. In a preferred embodiment ofthis aspect the compound is selected from among the compoundsspecifically disclosed herein.

[0054] In another aspect the invention features a method of eliciting asomatostatin receptor agonist effect in a subject in need thereof,wherein said method comprises administering to said subject an effectiveamount of a compound according to formula (I) or formula (II), or apharmaceutically acceptable salt thereof. In a preferred embodiment ofthis aspect the compound is selected from among the compoundsspecifically disclosed herein.

[0055] In another aspect the invention features a method ofsimultaneously eliciting both a dopamine receptor agonist effect and asomatostatin receptor agonist effect in a subject in need thereof,wherein said method comprises administering to said subject an effectiveamount of a compound according to formula (I) or formula (II), or apharmaceutically acceptable salt thereof. In a preferred embodiment ofthis aspect the compound is selected from among the compoundsspecifically disclosed herein.

[0056] In another aspect the invention features a pharmaceuticalcomposition comprising an effective amount of a compound according toformula (I) or formula (II), or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier. In a preferredembodiment of this aspect the compound is selected from among thecompounds specifically disclosed herein.

[0057] In another aspect the invention features a method of treating adisease or condition in a subject, said method comprising administeringto said subject a therapeutically effective amount of a compound offormula (I) or formula (II), or a pharmaceutically acceptable saltthereof, wherein said disease is selected from the list consisting oflung cancer, glioma, anorexia, hypothyroidism, hyperaldosteronism, H.pylori proliferation, acromegaly, restenosis, Crohn's disease, systemicsclerosis, external and internal pancreatic pseudocysts and ascites,VIPoma, nesidoblastosis, hyperinsulinism, gastrinoma, Zollinger-EllisonSyndrome, diarrhea, AIDS related diarrhea, chemotherapy relateddiarrhea, scleroderma, Irritable Bowel Syndrome, pancreatitis, smallbowel obstruction, gastroesophageal reflux, duodenogastric reflux,Cushing's Syndrome, gonadotropinoma, hyperparathyroidism, Graves'Disease, diabetic neuropathy, Paget's disease, polycystic ovary disease,thyroid cancer, hepatome, leukemia, meningloma, cancer cachexia,orthostatic hypotension, postprandial hypotension, panic attacks, GHsecreting adenomas, Acromegaly, TSH secreting adenomas, prolactinsecreting adenomas, insulinoma, glucagonoma, diabetes mellitus,hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy,proliferative retinopathy, dawn phenomenon, Nephropathy, gastric acidsecretion, peptic ulcers, enterocutaneous fistula, pancreaticocutaneousfistula, Dumping syndrome, watery diarrhea syndrome, pancreatitis,gastrointestinal hormone secreting tumor, angiogenesis, arthritis,allograft rejection, graft vessel bleeding, portal hypertension,gastrointestinal bleeding, obesity, and opioid overdose. In a preferredembodiment of this aspect the compound is selected from among thecompounds specifically disclosed herein. In a more preferred embodimentof this aspect of the invention said disease or condition is acromegaly.

[0058] In a particularly preferred embodiment of each of the foregoingmethods the compound is selected from the list of compounds consistingof Compound A through Compound K, or from among the list of compoundsconsisting of Example L through Example V, as disclosed hereinbelowunder the heading “Synthesis of Somatostatin-Dopamine Chimers”.

[0059] In another aspect of the invention is featured a dopamine agonistaccording the formula (I) or formula (II), hereinabove, wherein thesomatostatin analog “z” is replaced by a moiety comprising —H, —OH,(C₁-C₆)alkoxy, arylalkoxy, (e.g., benzyl, substituted benzyl, and thelike), —NH₂, —NR9R10, where R9 and R10 are as defined in formula (II).In a preferred embodiment of this aspect said dopamine agonist isselected from among the dopamine moiety components of thedopamine-somatostatin chimers disclosed herein, or a pharmaceuticallyacceptable salt thereof. In a most preferred embodiment of this aspectsaid dopamine agonist is:

[0060] or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0061] It is believed that one skilled in the art can, based on thedescription herein, utilise the present invention to its fullest extent.The following specific embodiments are, therefore, to be construed asmerely illustrative, and not limitative of the remainder of thedisclosure in any was whatsoever.

[0062] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Also, allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference, each in its entirety.

[0063] Various somatostatin receptors (SSTR's) have been isolated, e.g.,SSTR-1, SSTR-2, SSTR-3, SSTR4, and SSTR-5. Thus, a somatostatin agonistmay be one or more of an SSTR-1 agonist, SSTR-2 agonist, SSTR-3 agonist,SSTR-4 agonist or a SSTR-5 agonist. What is meant by, e.g., asomatostatin type-2 receptor agonist (i.e., SSTR-2 agonist) is acompound which has a high binding affinity (e.g., Ki of less than 100nM, or preferably less than 10 nm, or more preferably less than 1 nM)for SSTR-2 (e.g., as defined by the receptor binding assay describedbelow). What is meant by, e.g., a somatostatin type-2 receptor selectiveagonist is a somatostatin type-2 receptor agonist which has a higherbinding affinity (i.e., lower Ki) for SSTR-2 than for any othersomatostatin receptor.

[0064] In one embodiment the SSTR-2 agonist is also a SSTR-2 selectiveagonist. Examples of SSTR-2 agonists which may be used to practice thepresent invention include, but are not limited to:

[0065] D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;

[0066] cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe];

[0067]4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂;and

[0068]4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂.

[0069] Further examples of somatostatin agonists are those covered byformulae or those specifically recited in the publications set forthbelow, each of which is hereby incorporated by reference in itsentirety.

[0070] EP Application No. P5 164 EU (Inventor: G. Keri);

[0071] Van Binst, G. et al. Peptide Research 5:8 (1992);

[0072] Horvath, A. et al. Abstract, “Conformations of SomatostatinAnalogs Having Antitumor Activity”, 22nd European peptide Symposium,Sep. 13-19, 1992, Interlaken, Switzerland;

[0073] PCT Application No. WO 91/09056 (1991);

[0074] EP Application No. 0 363 589 A2 (1990);

[0075] U.S. Pat. No. 4,904,642 (1990);

[0076] U.S. Pat. No. 4,871,717 (1989);

[0077] U.S. Pat. No. 4,853,371 (1989);

[0078] U.S. Pat. No. 4,725,577 (1988);

[0079] U.S. Pat. No. 4,684,620 (1987);

[0080] U.S. Pat. No. 4,650,787 (1987);

[0081] U.S. Pat. No. 4,603,120 (1986);

[0082] U.S. Pat. No. 4,585,755 (1986);

[0083] EP Application No. 0 203 031 A2 (1986);

[0084] U.S. Pat. No. 4,522,813 (1985);

[0085] U.S. Pat. No. 4,486,415 (1984);

[0086] U.S. Pat. No. 4,485,101 (1984);

[0087] U.S. Pat. No. 4,435,385 (1984);

[0088] U.S. Pat. No. 4,395,403 (1983);

[0089] U.S. Pat. No. 4,369,179 (1983);

[0090] U.S. Pat. No. 4,360,516 (1982);

[0091] U.S. Pat. No. 4,358,439 (1982);

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[0094] U.S. Pat. No. 4,310,518 (1982);

[0095] U.S. Pat. No. 4,291,022 (1981);

[0096] U.S. Pat. No. 4,238,481 (1980);

[0097] U.S. Pat. No. 4,235,886 (1980);

[0098] U.S. Pat. No. 4,224,199 (1980);

[0099] U.S. Pat. No. 4,211,693 (1980);

[0100] U.S. Pat. No. 4,190,648 (1980);

[0101] U.S. Pat. No. 4,146,612 (1979);

[0102] U.S. Pat. No. 4,133,782 (1979);

[0103] U.S. Pat. No. 5,506,339 (1996);

[0104] U.S. Pat. No. 4,261,885 (1981);

[0105] U.S. Pat. No. 4,728,638 (1988);

[0106] U.S. Pat. No. 4,282,143 (1981);

[0107] U.S. Pat. No. 4,215,039 (1980);

[0108] U.S. Pat. No. 4,209,426 (1980);

[0109] U.S. Pat. No. 4,190,575 (1980);

[0110] EP Patent No. 0 389 180 (1990);

[0111] EP Application No. 0 505 680 (1982);

[0112] EP Application No. 0 083 305 (1982);

[0113] EP Application No. 0 030 920 (1980);

[0114] PCT Application No. WO 88/05052 (1988);

[0115] PCT Application No. WO 90/12811 (1990);

[0116] PCT Application No. WO 97/01579 (1997);

[0117] PCT Application No. WO 91/18016 (1991);

[0118] U.K. Application No. GB 2,095,261 (1981); and

[0119] French Application No. FR 2,522,655 (1983).

[0120] Note that for all somatostatin agonists described herein, eachamino acid residue represents the structure of —NH—C(R)H—CO—, in which Ris the side chain (e.g., CH₃ for Ala). Lines between amino acid residuesrepresent peptide bonds which join the amino acids. Also, where theamino acid residue is optically active, it is the L-form configurationthat is intended unless D-form is expressly designated. For clarity,disulfide bonds (e.g., disulfide bridge) which exist between two freethiols of Cys residues are not shown. Abbreviations of the common aminoacids are in accordance with IUPAC-IUB recommendations.

Synthesis of Somatostatin Acionists

[0121] The methods for synthesizing peptide somatostatin agonists arewell documented and are within the ability of a person of ordiniaryskill in the art. For example, peptides are synthesized on Rink amideMBHA resin(4-(2′4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHAresin) using a standard solid phase protocol of Fmoc chemistry. Thepeptide-resin with free amino functional at the N-terminus is thentreated with the corresponding compound containing dopamine moiety. Thefinal product is cleaved off from resin withTFA/water/triisopropylsilane (TIS) mixture.

[0122] For example, synthesis ofH-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH₂, can be achieved by followingthe protocol set forth in Example I of European Patent Application 0 395417 A1. The synthesis of somatostatin agonists with a substitutedN-terminus can be achieved, for example, by following the protocol setforth in PCT Publication No. WO 88/02756, PCT Publication No. WO94/04752, and/or European Patent Application No. 0 329 295.

[0123] Peptides can be and were cyclized by using iodine solution inMeOH/water and purified on C18 reverse-phase prep. HPLC, usingacetonitrile-0.1% TFA/water-0.1% TFA buffers. Homogeneity was assessedby analytical HPLC and mass spectrometry and determined to be >95% foreach peptide.

[0124] Certain uncommon amino acids were purchased from the followingvendors: Fmoc-Doc-OH and Fmoc-AEPA were purchased from Chem-ImpexInternational, Inc. (Wood Dale, Ill., USA). Fmoc-Caeg(Bhoc)-OH waspurchased from PerSeptive Biosystems (Framingham Mass., USA). Bhocstands for benzhydryloxycarbonyl.

Synthesis of Dopamine Agonists

[0125] The methods for synthesizing many dopamine agonists are also welldocumented and are within the ability of a person of ordinary skill inthe art Further synthetic procedures are provided in the followingreaction schemes and examples.

[0126] Similarly for compounds 6, 7 and 8:

[0127] Synthesis of Somatostatin-Dopamine Chimers

[0128] The somatostatin-dopamine chimers may be synthesized according tothe following reaction schemes and examples. Starting material andintermediates for compounds (I), (II) and (III), depicted in Scheme I,II, and III, respectively, are commercially available or prepared by theliteratures; Pharmazie 39, 537 (1984); collect Czech. Chem. Commun. 33,577 (1966); Helv. Chim. Acta 32, 1947, (1949)′ U.S. Pat. No. 5,097,031;U.S. Pat. No. 3,901,894; EP 0003667; U.S. Pat. No. 4,526,892. Thesynthesis of peptides are within the scope of a skilled person in theart, and in any event, is readily available in the literature. See,e.g., Stewart et al., Solid Phase Synthesis, Pierce Chemical, 2^(nd) Ed.1984; G. A. Grant; Synthetic peptide. W H., Freenand Co., New York,1992; M. Bodenszky A. Bodanszky, The Practice of Peptide Synthesis.Spring Venlag. N.Y. 1984.

[0129] Preparation of Compound A:

[0130] Compound 8 (3 eq.) is mixed withH-(Doc)₃-D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Abu-Cys(Acm)-Thr(tBu)-Rinkamide MBHA resin (1 eq.), HBTU (2.9 eq), HOBt (3.0 eq.) and DIEA (6 eq)in DMF. The mixture is shaken at room temperature for 4 hours. The resinis washed with DMF and DCM and dried under reduced pressure to dryness.The dry resin is treated with TFA/TIS/water (92/5/3, v/v) for 1 hour atroom temperature. The solution is filtered and concentrated. To theconcentrated solution is added cold ether. The precipitate is collectedand dissolved in water-methanol solvent system. To the solution is addediodine solution in methanol until the brown color appears. The solutionthen stands at room temperature for 1 hour. To the solution is addedNa₂S₂O₃ aqueous solution until the brown color disappears. The resultingsolution is purified by using a C18 reverse-phase prep HPLC, elutingwith a linear gradient of buffer A (1% TFA in Water)/buffer B (1% TFA inCH₃CN). The fractions are checked by analytical HPLC. The fractionscontaining pure desired compound are pooled and lyophilized to dryness.The molecular weight of the compound is measured by using MS fitted withan electrospray source.

[0131] Preparation of Compound B:

[0132] Compound 12 where R1 is n-propyl (1.5 eq.) is mixed withH-D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Abu-Cys(Acm)-Thr(tBu) Rinkamide MBHA resin (1 eq) and DIEA (2 eq) in DMF. The mixture is shaken atroom temperature for 5 hours. The resin is washed with DMF and DCM anddried under reduced pressure to dryness. The dry resin is treated withTFA/TIS/water (92/5/3, v/v) for 1 hour at room temperature. The solutionis filtered and concentrated. To the concentrated solution is added coldether. The precipitate is collected and dissolved in water-methanolsolvent system. To the solution is added iodine solution in methanoluntil the brown color appears. The solution then stands at roomtemperature for 1 hour. To the solution is added Na₂S₂O₃ aqueoussolution until the brown color disappears. The resulting solution ispurified by using a C18 reverse-phase prep HPLC, eluting with a lineargradient of buffer A (1% TFA in Water)/buffer B (1% TFA in CH₃CN). Thefractions are checked by analytical HPLC. The fractions containing puredesired compound are pooled and lyophilized to dryness. The molecularweight of the compound is measured by using MS fitted with anelectrospray source.

[0133] Preparation of Compound C:

[0134] Compound 11 where R1 is n-propyl (1.5 eq.) is mixed withH-AEPA-D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Abu-Cys(Acm)-Thr(tBu)-Rinkamide MBHA resin (1 eq) and DIEA (2 eq) in DMF. The mixture is shaken atroom temperature for 5 hours. The resin is washed with DMF and DCM anddried under reduced pressure to dryness. The dry resin is treated withTFA/TIS/water (92/5/3, v/v) for 1 hour at room temperature. The solutionis filtered and concentrated. To the concentrated solution is added coldether. The precipitate is collected and dissolved in water-methanolsolvent system. To the solution is added iodine solution in methanoluntil the brown color appears. The solution then stands at roomtemperature for 1 hour. To the solution is added Na₂S₂O₃ aqueoussolution until the brown color disappears. The resulting solution ispurified by using a C18 reverse-phase prep HPLC, eluting with a lineargradient of buffer A (1% TFA in Water)/buffer B (1% TFA in CH₃CN). Thefractions are checked by analytical HPLC. The fractions containing puredesired compound are pooled and lyophilized to dryness. The molecularweight of the compound is measured by using MS fitted with anelectrospray source.

[0135] Preparation of Compound D:

[0136] Compound 25 (3 eq.) is mixed withH-Doc-D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Abu-Cys(Acm)-Thr(tBu)-Rinkamide MBHA resin (1 eq.), HBTU (2.9 eq), HOBt (3.0 eq.) and DIEA (6 eq)in DMF. The mixture is shaken at room temperature for 4 hours. The resinis washed with DMF and DCM and dried under reduced pressure to dryness.The dry resin is treated with TFA/TIS/water (92/5/3, v/v) for 1 hour atroom temperature. The solution is filtered and concentrated. To it isadded cold ether, the precipitate is collected and dissolved inwater-methanol solvent system. To the solution is added iodine solutionin methanol until the brown color appears. The solution is then standsat room temperature for 1 hour. To the solution is added Na₂S₂O₃ aqueoussolution until the brown color disappears. The resulting solution ispurified by using a C18 reverse-phase prep HPLC, eluting with a lineargradient of buffer A (1% TFA in Water)/buffer B (1% TFA in CH₃CN). Thefractions are checked by analytical HPLC. The fractions containing puredesired compound are pooled and lyophilized to dryness. The molecularweight of the compound is measured by using MS fitted with anelectrospray source.

[0137] Preparation of Compound E:

[0138] Compound 26 (3 eq.) is mixed withH-(D-Ser(tBu))₅-Lys(Boc)-D-Tyr(tBu)-D-Tyr(tBu)-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Acm)-Trp(Boc)-Rinkamide MBHA resin (1 eq.), HBTU (2.9 eq), HOBt (3.0 eq.) and DIEA (6 eq)in DMF. The mixture is shaken at room temperature for 4 hours. The resinis washed with DMF and DCM and dried under reduced pressure to dryness.The dry resin is treated with TFA/TIS/water (92/5/3, v/v) for 1 hour atroom temperature. The solution is filtered and concentrated. To theconcentrated solution is added cold ether. The precipitate is collectedand dissolved in water-methanol solvent system. To the solution is addediodine solution in methanol until the brown color appears. The solutionthen stands at room temperature for 1 hour. To the solution is addedNa₂S₂O₃ aqueous solution until the brown color disappears. The resultingsolution is purified by using a C18 reverse-phase prep HPLC, elutingwith a linear gradient of buffer A (1% TFA in Water)/buffer B (1% TFA inCH₃CN). The fractions are checked by analytical HPLC. The fractionscontaining pure desired compound are pooled and lyophilized to dryness.The molecular weight of the compound is measured by using MS fitted withan electrospray source.

[0139] Preparation of Compound F:

Ethyl-[6-methyl-8β-ergolinylmethyl]thioacetate

[0140] To a solution of dihydrolysergol (240 mg) in 10 ml pyridine wasadded 250 μl methanesulforyl chloride. After stirring at roomtemperature for 2 hours, the reaction mixture was poured into 100 mlwater, it was extracted with chloroform (2×20 ml). Organic layer waswashed with water, then dried over MgSO₄ and solvent was removed invacuo to dryness to give 140 mg of pale brown solid. Further extractionfrom aqueous solution after basificabon with NaHCO₃ gave another 100 mgof product. Overall 240 mg. Mass Spec (Electrospray) 335.2.

[0141] To a solution of the above D-6-methyl-8β-mesyloxymethyl-ergoline(140 ng) in 3 ml dimethylformide was added powdered K₂CO₃ (150 mg)followed by 150 μl ethyl-2-mercaptoacetate and the mixture was heated at40° C. for 2 hours under nitrogen atmosphere. Solvent was removed invacuo to dryness, and the residue partitioned between chloroform andwater. Organic layer was then dried (MgSO₄), and after evaporation ofsolvent the residue was subject to preparative silica gel thin layerchromatography using chloroform/methanol (9:1) as developing solvents.Appropriate portion was isolated, extracted with chloroform-methanol andsolvents were removed in vacuo to dryness. Pale brown solid. 100 mg Massspec (Electrospray) 359.2.

[0142] Preparation of Compound G:

6-Methyl-8β-ergolinylmethylthioacetyl-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂

[0143] To a solution of 6-Methyl-8β-ergolimylmethylthioacetyl acid(Scheme I, compound 7) (50 mg) andD-Phe-c(Cys-Tyr(OBT)-D-Trp-Lys(BOC)-Abu-Cys)-Thr-NH₂ (100 mg) preparedby solid-phase synthesis using Fmoc-chemistry In 10 ml dimethylformidewas added 200 mg of EDC(1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide-HCL), 100 mg of HOAT(1-Hydroxy-7-azabezotriazole) followed by 200 μl diusopropyletylamineand the mixture was stirred at room temperature overnight. Volatilesubstances were removed in vacuo to dryness. The residue was partitionedbetween chloroform methanol and brine. The organic layer was washed withaqueous NaHCO₃, dried over MgSO₄. After evaporation of solvent, theresidue was subject to preparative thin-layer chromatography usingchloroform-methanol (85:15) as developing solvents. Appropriate portionwas isolated, extracted with chloroform-methanol and solvents wereremoved in Vacuo to give 40 mg of protected product. Mass Spec.(Electrospray) 1500.7.

[0144] The protected product was then treated with 30% trifluoroaceticacid in dichloromethal (10 ml) containing a few drops of triisopropylsiliane for 30 minutes. Volatile substances were removed in vacuo todryness. The residue was purified using vydac C₈ HPLC and CH₃CN/0.1%aqueous TFA, resulting in 17 mg of white solid. Mass Spec(Electrospray). 1344.8, 673.2.

[0145] Preparation of Compound H:

Ethyl-(6-n-propyl-8β-ergolinyl)methylthioacetate

[0146] This compound was prepared analogously to Compound F, startingwith D-n-propyl-8β-hydroxymethylergoline which can be made according toEP 000.667. Pale yellow solid. Mass Spec (Electropray) 387.2.

[0147] Preparation of Compound I:

6-n-propyl-8β-ergolinylmethylthioacetyl-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂

[0148] This compound was prepared analogously to Compound G, startingwith 6-n-propyl-8β-ergolinyl)methylthioacetic acid (Scheme I, compound6, where R1=propyl and s=1) andD-Phe-c(Cys-Tyr(OBT)-D-Trp-Lys(BOC)-Abu-Cys)-Thr-NH₂. White solid. MassSpec. (Electrospray) 1372.5, 687.3.

[0149] Preparation of Compound J:

6-D-Methyl-8β-ergolinylmethylthlaminosuccinoyl-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂

[0150] This compound was prepared analogously to Compound G startingwith 6-D-Methyl-8β-succinoylaminomethylergoline andD-Phe-c(Cys-Tyr(OBT)-D-Trp-Lys(BOC)-Abu-Cys)-Thr-NH₂, White solid. MassSpec (Electrospray) 1344.8, 673.2.

[0151] Preparation of Compound K:

[0152]6-allyl-8β-(1-ethyl-(3-N-methyl-3-carbonylmethyl)aminopropyl-ureidocarbonyl-ergoline-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2,i.e., a compound according to the following structure:

[0153] A.1-[[6-allylergolin-8β-yl]carbonyl]-1-[3-(N-(ethoxycarbonyl)methyl,N-methyl)amino-propyl]-3-ethylurea,i.e., a compound according to the following structure:

(1) 3,3-BOC, N-Methylpropanediamine

[0154] To a solution of 3 N-Methyl propanediamine (1.8 g) indichloromethane (30 ml) was added annhydrous MgSO4 (5.5 gm) followed bybenzaldehyde (2.3 g) and the mixture was stirred at room temperatureovernight. After filtration, the filtrate was treated with (BOC)₂ (4.3g) and DMAP (0.35 g) and stirred for about 1 hour. The mixture was thenwashed with 5% aqueous citric acid, then 5% NaHCO₃, and then dried overMgSO4.

[0155] After evaporation of solvent, the residue was dissolved inethanol (50 ml). Pd(OH)₂ (600 mg), acetic acid (1 ml), and cyclohexene(3 ml) were added and hydrogenation was carried out overnight. Themixture was filtered through a celite pad and the filtrate wasevaporated in vacuo to dryness to produce 3,3-BOC,N-Metyhlpropanediamineas a colorless liquid. 2.3 g. Mass Spec (Electrospray)=189.1.

(2) 6-allyl-8β-(3,3-BOC,N-Methyl-aminopropyl-carbamoyl)-ergoline

[0156] To a solution of 6-allyl-dihydrolysersic acid (150 mg), preparedaccording to the procedure disclosed in EP 0 003667, and3,3-BOC,N-Methyl-propanediamine (150 mg) in DMF (5 ml) was addeddiisopropylethylamine (175 μl ) followed by diethylcyanophosphonate (150μl) and the mixture was stirred at room temperature overnight. Volatilesubstances were removed in vacuo to dryness. The residue was partitionedbetween CHCl₃ and water. The organic layer was then washed with aqueousNaHCO₃ and dried over MgSO₄. Solvent was removed in vacuo to give6-allyl-8β-(3,3-BOC, N-Methyl-aminopropyl-carbamoyl)-ergoline.

(3) 6-allyl-8β-(3-N-Methyl-aminopropyl-carbamoyl)-ergoline, TFA salt

[0157] 6-allyl-8β-(3,3-BOC,N-Methyl-aminopropyl-carbamoyl)-ergoline fromthe previous step was treated with 30% TFA in dichloromethane for 30minutes and volatile substances were removed in vacuo to drynessyielding 250 mg of6-allyl-8β-(3-N-Methyl-aminopropyl-carbamoyl)-ergoline, TFA salt. Massspec (Electrospray)=367.2.

(4)6-allyl-8,β-(3-N-Methyl,3-carbethoxymethyl)aminopropyl-carbamoyl-ergoline

[0158] To a solution of6-allyl-8,β-(3-N-Methyl-aminopropyl-carbamoyl)-ergoline TFA salt (250mg) and K₂CO₃ (140 mg) in DMF (5 ml) was added ethyl bromoacetate (70μl) and the mixture was stirred at room temperature overnight. Afterevaporation of solvent, the residue was partitioned between chloroformand water. The organic layer was dried using MgSO₄ and then solvent wasremoved in vacuo to give crude6-allyl-8β-(3-N-Methyl,3-carbethoxymethyl)aminopropyl-carbamoyl-ergoline(240 mg). Mass Spec (Electrospray)=453.2.

(5)6-allyl-8β-(1-ethyl-(3-N-methyl-3-carbethoxymethyl)aminopropyl-ureidocarbonyl-ergoline

[0159]6-allyl-8’-(3-N-Methyl,3-carbethoxymethyl)aminopropylcarbamoyl-ergolinefrom the previous step was dissolved in toluene (10 ml) andethylisocyanate (3 ml) was added. The mixture was refluxed undernitrogen atmosphere for 3 days and after evaporation of volatilesubstances, the residue was subject to preparative silica gelchromatography using chloroform/methanol (19 to 1) as developingsolvents. Appropriate portion was extracted with chloroform/methanol andsolvents were removed in vacuo to give6-allyl-8β-(1-ethyl-(3-N-methyl-3-carbethoxymethyl)aminopropyl-ureidocarbonyl-ergoline as a pale yellow viscous substance(30 mg). Mass Spec (Electrospray)=524.3.

[0160] B.6-ally-8β-(1-ethyl-(3-N-methyl-3-carboxymethyl)aminopropyl-ureidocarbonyl-ergoline,i.e., a compound according to the following structure:

[0161] To a mixture of6-allyl-8β-(1-ethyl-(3-N-methyl-3-carbethoxymethyl)aminopropyl-ureidocarbonyl-ergoline(520 mg) in 10 ml of acetone are added 15 ml of 0.2M phosphate buffer(pH=approx. 7) and 0.6 ml ChiroCLEC-BL (Altus Biologics, Cambridge,Mass.). The mixture is incubated on a rotary shaker at approximately 40C overnight. The mixture is acidified with 5% aqueous citric acid andextracted with CHCl₃-Methanol. The organic extract is dried and thesolvents are removed in vacuo to yield6-allyl-8β-(1-ethyl-(3-N-methyl-3-carboxymethyl)aminopropyl-ureidocarbonyl-ergoline.

[0162] C.6-allyl-8β-(1-ethyl-(3-N-methyl-3-carbonylmethyl)aminopropyl-ureidocarbonyl-ergoline-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2,i.e., Compound K

[0163] To a solution of6-allyl-8β-(1-ethyl-(3-N-methyl-3-carboxymethyl)aminopropyl-ureidocarbonyl-ergoline(50 mg) and D-Phe-c(Cys-Tyr-D-Trp-Lys(FMOC)-Abu-Cys)-Thr-NH₂ (100 mg,prepared by solid-phase synthesis), in 10 ml dimethylformide is added200 mg of EDC (1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide-HCL),100 mg of HOAT (1-Hydroxy-7-azabezotriazole) followed by 200 μldiisopropyletylamine and the mixture is stirred at room temperatureovernight. Volatile substances are removed in vacuo to dryness. Theresidue is partitioned between chloroform methanol and brine. Theorganic layer is washed with aqueous NaHCO₃ and then dried over MgSO₄.After evaporation of solvent the protected product is then treated with5% piperidine in DMF (10 ml) for 30 minutes. Volatile substances areremoved in vacuo to a small volume (about 2 ml). It is purified usingVYDAC C₁₈ HPLC and CH₃CN/0.1% aqueous TFA to yield the purified,de-protected product.

EXAMPLE L

[0164]

[0165] A.H-Aepa-Lys(Boc)-DTyr(tBu)-DTyr(tBu)-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin

[0166] The protected peptide-resin was automatically synthesized on anApplied Biosystems (Foster City, Calif.) model 433A peptide synthesizerby using Fluorenylmethyloxycarbonyl (Fmoc) chemistry. A Rink Amide MBHAresin (Novabiochem., San Diego, Calif.) with substitution of 0.72 mmol/gwas used. The Fmoc amino acids (AnaSpec, San Jose, Calif.) were usedwith the following side chain protection: Fmoc-Thr(tBu)-OH,Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-DTrp(Boc)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-DTyr(tBu)-OH Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH andFmoc-Abu-OH. Fmoc-Aepa-OH was purchased from Chem-Impex International,Inc. (Wood Dale, Ill.). The synthesis was carried out on a 0.25 mmolscale. The Fmoc groups were removed by treatment with 20% piperidine inN-methylpyrrolidone (NMP) for 30 min. In each coupling step, the Fmocamino acid (4 eq, 1 mmol) was first pre-activated in 2 mL solution of0.45M 2-(1-H-benzotriazole-1-yl)-1,1,2,3-tetramethyluroniumhexafluorophosphate/1-hydroxy-benzotriazole (HBTU/HOBT) inN,N-dimethylformamide (DMF). This activated amino acid ester, 1 mL ofdiisopropylethylamine (DIEA) and 1 mL of NMP were added to the resin.The ABI 433A peptide synthesizer was programmed to perform the followingreaction cycle: (1) washing with NMP, (2) removing Fmoc protecting groupwith 20% piperidine in NMP for 30 min, (3) washing with NMP, (4)coupling with pre-activated Fmoc amino acid for 1 h. The resin wascoupled successively according to the sequence. After peptide chain wasassembled, the Fmoc was removed and washed completely by using DMF anddichloromethane (DCM).

[0167] MBHA=4-methylbenzyihydrylamine

[0168] B. The resultingH-Aepa-Lys(Boc)-DTyr(tBu)-DTyr(tBu)-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin (0.188 mmol) was mixed with compound 7 (92 mg, 0.28mmol, 1.5 eq.), ), [7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium-hexafluorophosphate] (PyAOP) (146 mg, 0.28mmol, 1.5 eq.) and 1-hydroxy-7-azabenzotriazol (HOAT) (38 mg, 0.28 mmol,1.5 eq.) in 5 mL of DCM. The mixture was shaken overnight. The resin wasdrained and washed successively with DMF, methanol and DCM. After dryingin the air, the resin was treated with a mixture of TFA, H₂O andtriisopropylsilane (TIS) (9.5 ml/0.85 ml/0.8 ml) for 2 h. The resin wasfiltered off and the filtrate was poured into 50 mL of cold ether. Theprecipitate was collected after centrifuge. The crude product wasdissolved in 100 ml of 5% AcOH aqueous solution, to which iodinemethanol solution was added dropwise until yellow color maintained. Thereaction solution was stirred for additional 1 h. 10% Na₂S₂O₃ watersolution was added to quench excess iodine. The crude product in thesolution was purified on preparative HPLC system with a column (4×43 cm)of C18 DYNAMAX-100 A⁰ (Varian, Walnut Creek, Calif.). The column waseluted with a linear gradient from 80% A and 20% B to 55% A and 45% B in50 min., where A was 0.1% TFA in water and B was 0.1% TFA inacetonitrile. The fractions were checked by an analytical HPLC. Thosecontaining pure product were pooled and lyophilized to dryness. Yield:40%. The purity was 96.8% based on analytical HPLC analysis. MS (ElectroSpray): 1820.8 (in agreement with the calculated molecular weight of1821.3).

EXAMPLE M

[0169]

[0170] Example M was synthesized substantially according to theprocedure described for Example L by usingH-Lys(Boc)-DTyr(tBu)-DTyr(tBu)-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 97.9% based onanalytical HPLC analysis. MS (Electro Spray): 1652.1 (in agreement withthe calculated molecular weight of 1652.03).

EXAMPLE N

[0171]

[0172] Example N was synthesized substantially according to theprocedure described for Example L by usingH-Doc-Lys(Boc)-DTyr(tBu)-DTyr(tBu)-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 99.2% based onanalytical HPLC analysis. MS (Electro Spray): 1797.1 (in agreement withthe calculated molecular weight of 1797.19).

[0173] Fmoc-Doc-OH was purchased from Chem-Impex International, Inc.(Wood Dale, Ill.).

EXAMPLE O

[0174]

[0175] Example O was synthesized substantially according to theprocedure described for Example L by using(6-N-propyl-8β-ergolinyl)methylthioacetic acid andH-Lys(Boc)-DTyr(tBu)-DTyr(tBu)-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 97.4% based onanalytical HPLC analysis. MS (Electro Spray): 1680.6 (in agreement withthe calculated molecular weight of 1680.1).

EXAMPLE P

[0176]

[0177] Example P was synthesized substantially according to theprocedure described for Example L by using(6-N-propyl-8β-ergolinyl)methylthioacetic acid andH-Aepa-Aepa-D-Phe-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 99.9% based onanalytical HPLC analysis. MS (Electro Spray): 1710.7 (in agreement withthe calculated molecular weight of 1711.2).

EXAMPLE Q

[0178]

[0179] Example Q was synthesized substantially according to theprocedure described for Example L by using(6-N-propyl-8β-ergolinyl)methylthioacetic acid andH-Aepa-Aepa-DPhe-Cys(Trt)-(3-lodo)Tyr-DTrp(Boc)-Lys(Boc)-Val-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 99% based onanalytical HPLC analysis. MS (Electro Spray): 1851.1 (in agreement withthe calculated molecular weight of 1851.1).

[0180] Fmoc-(3-lodo)-Tyr-OH was purchased from Advanced ChemTech(Louisville, Ky.).

EXAMPLE R

[0181]

[0182] Example R was synthesized substantially according to theprocedure described for Example L by usingH-Aepa-DPhe-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 98.3% based onanalytical HPLC analysis. MS (Electro Spray): 1513.8 (in agreement withthe calculated molecular weight of 1513.9).

EXAMPLE S

[0183]

[0184] Example S was synthesized substantially according to theprocedure described for Example L by usingH-Aepa-Aepa-DPhe-Cys(Trt)-(3-lodo)Tyr-DTrp(Boc)-Lys(Boc)-Val-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 85.7% based onanalytical HPLC analysis. MS (Electro Spray): 1822.9 (in agreement withthe calculated molecular weight of 1823.06).

EXAMPLE T

[0185]

[0186] Example T was synthesized substantially according to theprocedure described for Example L by usingH-Doc-DPhe-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 98.9% based onanalytical HPLC analysis. MS (Electro Spray): 1489.6 (in agreement withthe calculated molecular weight of 1489.84).

EXAMPLE U

[0187]

[0188] Example U was synthesized substantially according to theprocedure described for Example L by usingH-Doc-DPhe-Cys(Trt)-(3-lodo)Tyr-DTrp(Boc)-Lys(Boc)-Val-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. MS (Electro Spray): 1629.8 (in agreement with thecalculated molecular weight of 1629.7).

EXAMPLE V

[0189]

[0190] The titled compound was synthesized substantially according tothe procedure described for Example L by usingH-Doc-Doc-DPhe-Cys(Trt)-Tyr(tBu)-DTrp(Boc)-Lys(Boc)-Abu-Cys(Trt)-Thr(tBu)-RinkAmide MBHA Resin. Purity of the final product was 99% based onanalytical HPLC analysis. MS (Electro Spray): 1635.0 (in agreement withthe calculated molecular weight of 1633).

[0191] Some of the compounds of the instant invention can have at leastone asymmetric center. Additional asymmetric centers may be present onthe molecule depending upon the nature of the various substituents onthe molecule. Each such asymmetric center will produce two opticalisomers and it is intended that all such optical isomers, as separated,pure or partially purified optical isomers, racemic mixtures ordiastereomeric mixtures thereof, are included within the scope of theinstant invention.

[0192] The compounds of the instant invention generally can be isolatedin the form of their pharmaceutically acceptable acid addition salts,such as the salts derived from using inorganic and organic acids.Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric,formic, acetic, trifluoroacetic, propionic, maleic, succinic,D-tartaric, L-tartaric, malonic, methane sulfonic and the like. Inaddition, certain compounds containing an acidic function such as acarboxy can be isolated in the form of their inorganic salt in which thecounter-ion can be selected from sodium, potassium, lithium, calcium,magnesium and the like, as well as from organic bases.

[0193] The pharmaceutically acceptable salts can be formed by takingabout 1 equivalent of a compound of the invention, (e.g., Compound C,below), and contacting it with about 1 equivalent or more of theappropriate corresponding acid of the salt which is desired. Work-up andisolation of the resulting salt is well-known to those of ordinary skillin the art.

[0194] The compounds of this invention can be administered by oral,parenteral (e.g., intramuscular, intraperitoneal, intravenous orsubcutaneous injection, or implant), nasal, vaginal, rectal, sublingualor topical routes of administration and can be formulated withpharmaceutically acceptable carriers to provide dosage forms appropriatefor each route of administration. Accordingly, the present inventionincludes within its scope pharmaceutical compositions comprising, as anactive ingredient, at least one compound of the invention in associationwith a pharmaceutically acceptable carrier.

[0195] Solid dosage forms for oral administration include capsules,tablets, pills, powders and granules. In such solid dosage forms, theactive compound is admixed with at least one inert pharmaceuticallyacceptable carrier such as sucrose, lactose, or starch. Such dosageforms can also comprise, as is normal practice, additional substancesother than such inert diluents, e.g., lubricating agents such asmagnesium stearate. In the case of capsules, tablets and pills, thedosage forms may also comprise buffering agents. Tablets and pills canadditionally be prepared with enteric coatings.

[0196] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,the elixirs containing inert diluents commonly used in the art, such aswater. Besides such inert diluents, compositions can also includeadjuvants, such as wetting agents, emulsifying and suspending agents,and sweetening, flavoring and perfuming agents.

[0197] Preparations according to this invention for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, or emulsions. Examples of non-aqueous solvents or vehiclesare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil and corn oil, gelatin, and injectable organic esters such as ethyloleate. Such dosage forms may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. They may be sterilized by,for example, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

[0198] Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

[0199] Compositions for nasal or sublingual administration are alsoprepared with standard excipients well known in the art.

[0200] In general, an effective dosage of active ingredient in thecompositions of this, invention may be varied; however, it is necessarythat the amount of the active ingredient be such that a suitable dosageform is obtained. The selected dosage depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment, all of which are within the realm of knowledge of oneof ordinary skill in the art. Generally, dosage levels of between 0.0001to 100 mg/kg of body weight daily are administered to humans and otheranimals, e.g., mammals.

[0201] A preferred dosage range is 0.01 to 10.0 mg/kg of body weightdaily, which can be administered as a single dose or divided intomultiple doses.

[0202] Somatostatin Receptor Specificity and Selectivity Assay

[0203] Specificity and selectivity of the somatostatin analogues used tosynthesize the somatostatin-dopamine chimers were determined by aradioligand binding assay on CHO-K1 cells stably transfected with eachof the SSTR subtypes, as follows.

[0204] The complete coding sequences of genomic fragments of the SSTR 1,2, 3, and 4 genes and a cDNA clone for SSTR 5 were subcloned into themammalian expression vector pCMV (Life Technologies, Milano, Italy).Clonal cell lines stably expressing SSTR's 1-5 were obtained bytransfection into CHO-KI cells (ATCC, Manassas, Va., USA) using thecalcium phosphate co-precipitation method (Davis L, et al., 1994 In:Basic methods in Molecular Biology, 2nd edition, Appleton & Lange,Norwalk, Conn., USA: 611-646). The plasmid pRSV-neo (ATCC) was includedas a selectable marker. Clonal cell lines were selected in RPMI 1640media containing 0.5 mg/ml of G418 (Life Technologies, Milano, Italy),ring cloned, and expanded into culture.

[0205] Membranes for in vitro receptor binding assays were obtained byhomogenizing the CHO-K1 cells expressing the SSTR's subtypes in ice-cold50 mM Tris-HCl and centrifuging twice at 39000 g (10 min), with anintermediate resuspension in fresh buffer. The final pellets wereresuspended in 10 mM Tris-HCl for assay. For the SSTR 1, 3, 4, and 5assays, aliquots of the membrane preparations were incubated 90 min. at25° C. with 0.05 nM [¹²⁵I-Tyr11]SS-14 in 50 mM HEPES (pH 7.4) containing10 mg/ml BSA, 5 mM MgCl₂, 200 KIU/ml Trasylol, 0.02 mg/ml bacitracin,and 0.02 mg/ml phenylmethylsuphonyl fluoride. The final assay volume was0.3 ml. For the SSTR 2 assay, 0.05 nM [¹²⁵I]MK-678 was employed as theradloligand and the incubation time was 90 min at 25° C. The Incubationswere terminated by rapid filtration through GF/C filters (pre-soaked in0.3% polyethylenimine) using a Brandel filtration manifold. Each tubeand filter were then washed three times with 5 ml aliquots of ice-coldbuffer. Specific binding was defined as the total radioligand boundminus that bound in the presence of 1000 nM SS-14 for SSTR 1, 3, 4, and5, or 1000 nM MK-678 for SSTR2.

[0206] Dopamine Receptor Specificity and Selectivity Assay

[0207] Specificity and selectivity for the dopamine-2 receptor of thedopamine analogues used to synthesize the somatostatin-dopamine chimersmay be determined by a radioligand binding assay as follows.

[0208] Crude membranes were prepared by homogenization of frozen ratcorpus striatum (Zivic Laboratories, Pittsburgh, Pa.) in 20 ml ofice-cold 50 mM Tris-HCl with a Brinkman Polytron (setting 6, 15 sec).Buffer was added to obtain a final volume of 40 ml, and the homogenatewas centrifuged in a Sorval SS-34 rotor at 39,000 g for 10 min at 0-4°C. The resulting supernatant was decanted and discarded. The pellet wasrehomogenized in ice-cold buffer, pre-incubated at 37° C. for 10 min,diluted, and centrifuged as before. The final pellet was resuspended inbuffer and held on ice for the receptor binding assay.

[0209] For assay, aliquots of the washed membrane preparations and testcompounds were Incubated for 15 min (37 C) with 0.25 nM [³HI]spiperone(16.5 Ci.mmol, New England Nuclear, Boston, Mass.) in 50 mM Tris HCl,120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2. The final assay volumewas 1.0 ml. The incubations were terminated by rapid filtration throughGF/B filters using a Brandel filtration manifold. Each tube and filterwere then washed three times with 5-ml aliquots of ice-cold buffer.Specific binding was defined as the total radioligand bound minus thatbound in the presence of 1000 nM (+) butaclamol.

Other Embodiments

[0210] It is to be understood that while the invention has beendescribed In conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the claims.Also, all publications mentioned herein are hereby incorporated byreference in their entirety.

What is claimed is:
 1. A compound of the formula (I),

wherein: X is H, Cl, Br, I, F, —CN, , or C₁₋₅ alkyl; R1 is H, C₁₋₄alkyl, allyl, alkenyl or —CN; R2 and R3, each are, independently H orabsent, provided that when R2 and R3 are absent a double bond is presentbetween the carbon atoms to which they are attached; R4 is H or —CH₃; Yis —O—, —C(O)—, —S—, —S—(CH₂)s-C(O)—, —S(O)—, —S(O)₂—, —SC(O)—, —OC(O)—,—N(R5)-C(O)—, or —N(R6)—; R6, R7, R8, R9 and R10 each is, independently,H or C₁₋₅ alkyl; R6 is H or C₁₋₅ alkyl; m is 0 or 1; n is 0-10; L is—(CH₂)p-C(O)—, when Y is —S—, —S(O)—, —S(O)₂—, —O— or —N(R6)-; L is—C(O)—(CR7R8)q-C(O)—, when Y is —N(R6)-, —O—, or —S—; L is -(Doc)t-,when Y is —C(O)—, SC(O)—, —OC(O)—, —S—(CH₂)s-C(O)—, or —N(R5)-C(O)—; pis 1-10; q is 2-4; s is 1-10; t is 1-10; and Z is somatostatin analog ora moiety comprising —H, —OH, (C₁-C₆)alkoxy, arylalkoxy, —NH₂, or—NR9R10, wherein R9 and R10 each is, independently, H or C₁₋₅ alkyl; ora pharmaceutically acceptable salt thereof.
 2. A compound of the formula(II),

wherein: X is H, Cl, Br, I, F, —CN, , or C₁₋₅ alkyl; R1 is C1-4 alkyl,H, allyl, alkenyl or —CN; R2 and R3, each are, independently H orabsent, provided that when R2 and R3 are absent a double bond is presentbetween the carbon atoms to which they are attached; R4 is H or —CH₃; R5is C1-5 alkyl group, or a group of the formula of —(CH₂)rN(CH₃)q; Y is—O—, —C(O) , —S—, —SC(O)—, —OC(O)—, —N(R6)-C(O)—, —N(R7)-, or—N(R8)-(CH₂)s-C(O)—; R6, R7, R8, R9 and R10 each is, independently, H orC₁₋₅ alkyl; L is —(CH₂)p-C(O)—, when Y is —S—, —O— or —N(R7)-; L is—C(O)—(CR9R10)q-C(O)—, when Y is —N(R7)-, —O—, or —S—; L is -(Doc)t-,when Y is —C(O)—, SC(O)—, —OC(O)—, —N(R8)-(CH2)s-C(O)—, or —N(R6)-C(O)—;m is 0 or 1; n is 2-10; r is 1-8; q is 2-4; p is 1-10; s is 1-10; t is1-10; and Z is somatostatin analog or a moiety comprising —H, —OH,(C₁-C₆)alkoxy, arylalkoxy, —NH₂, or —NR9R10; or a pharmaceuticallyacceptable salt thereof.
 3. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 4. A compound accordingto the formula:

or a pharmaceutically acceptable salt thereof.
 5. A compound accordingto the formula:

Ethyl-[6-methyl-8β-ergolinylmethyl]thioacetate;6-Methyl-8β-ergolinylmethylthloacetyl-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂;Ethyl-(6-n-propyl-8β-ergolinyl)methylthioacetate;6-n-propyl-8β-ergolinylmethylthioacetyl-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂;6-D-Methyl-8β-ergolinylmethylthlaminosuccinoyl-D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH₂;

or a pharmaceutically acceptable salt thereof.
 6. A compound accordingto the formula:

or a pharmaceutically acceptable salt thereof.
 7. A method of elicitinga dopamine receptor agonist effect in a subject in need thereof, whereinsaid method comprises administering to said subject an effective amountof a compound according to any one of claims 1-6, or a pharmaceuticallyacceptable salt thereof.
 8. A method of eliciting a somatostatinreceptor agonist effect in a subject in need thereof, wherein saidmethod comprises administering to said subject an effective amount of acompound according to any one of claims 1-6, or a pharmaceuticallyacceptable salt thereof.
 9. A method of simultaneously eliciting both adopamine receptor agonist effect and a somatostatin receptor agonisteffect in a subject in need thereof, wherein said method comprisesadministering to said subject an effective amount of a compoundaccording to any one of claims 1-6, or a pharmaceutically acceptablesalt thereof.
 10. A pharmaceutical composition comprising an effectiveamount of a compound according to any one of claims 1-6, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 11. A method of treating a disease in a subject,said method comprising administering to said subject a therapeuticallyeffective amount of a compound according to any one of claims 1-6,wherein said disease is selected from the list consisting of lungcancer, glioma, anorexia, hypothyroidism, hyperaldosteronism, H. pyloriproliferation, acromegaly, restenosis, Crohn's disease, systemicsclerosis, external and internal pancreatic pseudocysts and ascites,VIPoma, nesidoblastosis, hyperinsulinism, gastrinoma, Zollinger-EllisonSyndrome, diarrhea, AIDS related diarrhea, chemotherapy relateddiarrhea, scleroderma, Irritable Bowel Syndrome, pancreatitis, smallbowel obstruction, gastroesophageal reflux, duodenogastric reflux,Cushing's Syndrome, gonadotropinoma, hyperparathyroidism, Graves'Disease, diabetic neuropathy, Paget's disease, polycystic ovary disease,thyroid cancer, hepatome, leukemia, meningioma, cancer cachexia,orthostatic hypotension, postprandial hypotension, panic attacks, GHsecreting adenomas, Acromegaly, TSH secreting adenomas, prolactinsecreting adenomas, insulinoma, glucagonoma, diabetes mellitus,hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy,proliferative retinopathy, dawn phenomenon, Nephropathy, gastric acidsecretion, peptic ulcers, enterocutaneous fistula, pancreaticocutaneousfistula, Dumping syndrome, watery diarrhea syndrome, pancreatitis,gastrointestinal hormone secreting tumor, angiogenesis, arthritis,allograft rejection, graft vessel bleeding, portal hypertension,gastrointestinal bleeding, obesity, and opioid overdose.
 12. The methodaccording to claim 11, wherein said disease or condition is acromegally.13. A method according to any one of claims 7, 8, 9, 11 or 12, whereinsaid compound is

or a pharmaceutically acceptable salt thereof.
 14. A pharmaceuticalcomposition according to claim 10, wherein said compound is:

or a pharmaceutically acceptable salt thereof.
 15. A compound accordingto claim 1 or 2, wherein z is a moiety comprising —H, —OH,(C₁-C₆)alkoxy, arylalkoxy, —NH₂, or —NR9R10; or a pharmaceuticallyacceptable salt thereof.
 16. A compound according to claim 15, wherein zis a moiety comprising —H, —OH, (C1-C6)alkoxy, or benzyl; or apharmaceutically acceptable salt thereof.
 17. A compound according toclaim 15, wherein said compound is according to the formula:

or a pharmaceutically acceptable salt thereof.